Method for detecting a substance and microtiter plate

ABSTRACT

The invention relates to a method for detecting an analyte A in a liquid, comprising the following steps: (a) a solution containing an analytical reagent N is provided in a container B; (b) the analyte A is added to the solution, (c) an electrical field eF which acts upon the solution is applied by means of electrodes disposed outside the container B, whereby a modification occurs in the concentration of a substance which is specific with respect to the presence of the analyte A in a region M of the container B and (d) the modification of said concentration is optically detected.

[0001] The invention relates to a method for detecting an analyte, andto a microtiter plate suitable therefor.

[0002] The invention relates generally to the area of looking for anddetecting pharmacological active ingredients. It is known in the art tobring a test substance or substance into contact with a plurality ofdifferent potential reactants for example in a microtiter plate.

[0003] If the substance has an affinity for a potential reactant, areaction takes place between the substance and the reactant. Thereaction may be, for example, a chemical conversion or a binding. Thereaction is detected by means of a change in the physical properties ofthe solution.

[0004] Detection methods used are for instance fluorescencepolarization, fluorescence resonance energy transfer, fluoresencecolleration spectroscopy and radiolabeling methods. The known detectionmethods are complicated. In some cases they require the use of poisonoussubstances.

[0005] Also known in the art are methods which enable a plurality ofsubstance mixtures to be separated in parallel by electrophoresis. Forexample, Advanced Biotechnologies offers under the sign “Midge” anapparatus with which electrophoretic separation of up to 100 DNA samplesin parallel is possible. For this purpose, each DNA sample is felt intoa well of a gel. An electric field is then applied across the gel sothat the samples are transported into the gel and fractionated there.Evaluation takes place via the distance the constituents of the DNAsample have migrated in the gel. The known method is time-consuming. DE199 52 160 A1 discloses a method for detecting a first molecule in asolution, in which a dye-coupled second molecule can bind to the firstmolecule. The net electric charge on the second molecule is in this casesmaller in size than and of opposite sign to the net electric charge onthe first molecule. An anode and a cathode are present in the solution,and an electric field is applied between them. The dye is detectedoptically at the anode if the net electric charge of the first moleculeis negative, and at the cathode if the net electric charge of the firstmolecule is positive. The method requires a complicated measuringapparatus in which a light beam must be focused exactly on the electrodein order to be able to detect the optical change at the electrode.

[0006] It is an object of the invention to eliminate the prior artdisadvantages. It is particularly intended to indicate a method and amicrotiter plate with which it is possible universally and without greateffort simultaneously to investigate the reaction behavior of aplurality of substances.

[0007] This object is achieved by the features of claims 1 and 14.Expedient refinements of the invention are evident from the features ofclaims 2 to 13 and 15 to 25.

[0008] According to the invention, a method for detecting a substance isprovided having the following steps:

[0009] a) provision of a solution comprising a detection reagent in acontainer,

[0010] b) addition of the analyte to the solution,

[0011] c) application of an electric field acting on the solution bymeans of electrodes located outside the container, so that theconcentration of an entity which is specific for the presence of theanalyte changes in a region of the container, and

[0012] d) optical detection of the concentration change.

[0013] The proposed method is universal. It is simple to carry out. Theuse of hazardous or poisonous detection reagents is unnecessary. It ispossible in particular to investigate simultaneously the effect of alarge number of detection reagents on one analyte. The detection reagentis specific for the analyte. It reacts or binds with the analyte so thatits electrophoretic mobility changes. It is particularly advantageousthat, apart from the addition of the analyte in step b, no furtheraddition to or removal from the container is necessary. It is possiblethereby to avoid pipetting errors, and a high sample throughput is madepossible. Since the electrodes are located outside the container it ispossible to avoid electrolytic reactions at the electrodes. A furtheradvantage is that exact focusing of a light beam on a predeterminedpoint of the container is unnecessary for detection of the concentrationchange. The apparatus for optical detection of the concentration changecan therefore have a simpler construction than an apparatus fordetecting optical changes at an electrode.

[0014] The solution can be incubated after step b. The container,especially its wall or its base, preferably consists at leastsectionally of an ion-conducting material and the electric field (eF) isapplied in step c in such a way that a migration of ions in the materialis brought about thereby. It is thus possible to achieve a concentrationchange specific for the analyte and optically detectable. The ions canmoreover migrate out of the liquid into the material, out of thematerial into the liquid or through the material. No removal of liquidfrom the container is necessary for the optical detection of theconcentration change in step d. The optical detection expediently takesplace through the opening and/or the base of the container.

[0015] The entity is advantageously a detection reagent, a reactionproduct formed from the detection reagent and the substance, or acompetitor. The detection reagent may additionally comprise a receptor,a competitor or a precursor of the reaction product. The receptor isexpediently selected from the following group: peptide, protein, nucleicacid, sugar, antibody, lectin, avidin, streptavidin, PNA (peptidenucleic acid) or LNA (locked nucleic acid).

[0016] The entity may be labeled with a fluorophore. A possible exampleis a molecular beacon. The entity may be bound onto and/or in the baseof the container.

[0017] For the optical detection of the concentration change, a lightbeam is passed at least through the region, and the change thereofbrought about by the entity is measured. One possible light beam is alaser beam. Transmitted or reflected light can be utilized. The changebrought about in the light beam may be a change in intensity, a changein the plane of polarization, a scattering angle or the like. The lightbeam is expediently guided so that it enters or emerges substantiallyperpendicularly from the base or the opening of the container. Thissimplifies the measurement. The method is thus suitable for standardmicrotiter plate readers.

[0018] In a particularly advantageous embodiment, the electric field isapplied simultaneously across a plurality of containers. The opticaldetection of the concentration change in the plurality of containers canlikewise take place simultaneously. The containers are expedientlycontainers arranged in the manner of a microtiter plate on a commonsupport.

[0019] Further according to the invention for carrying out the method amicrotiter plate is provided with a plurality of containers which areformed at least sectionally of an ion-conducting material. The use of anion-conducting material makes it possible to bring about, through amigration of ions which is caused in the electric field, a concentrationchange which is specific for the analyte and which can be detectedoptically.

[0020] It is expedient for the walls and/or the base of the containersto be produced from the ion-conducting material. The base may beproduced from an electrical insulator. It may also be activated for thebinding of a ligand, receptor or substrate. However, it is also possiblefor a receptor or substrate to be immobilized on the base. In a furtherembodiment, different receptors can be immobilized on predeterminedsections of the base. The base may be produced from glass, quartz orplastic.

[0021] The walls may consist of a porous material. They may be activatedfor the binding of a ligand. It is additionally possible for the wallsto comprise auxiliaries, e.g. quenchers or protein- or nucleicacid-binding entities.

[0022] The containers may, according to a further embodiment feature,have a substantially rectangular cross section. In this case two wallsof the container are arranged parallel to the electrodes. With such anarrangement, the electric field is developed homogeneously over thevolume of the container.

[0023] The ion-conducting material may be produced from a material whichis preferably selected from the following group: agarose,polyacrylamide, cellulose, paper, paperboard, porous silicate,polystyrene, polyvenyl chloride, polycarbonate, nylon, polyethylene.Other materials with ion-conducting properties are of course alsosuitable. The aforementioned materials are expediently in porous form.

[0024] In a particularly advantageous embodiment, the containers are inthe form of recesses in a first plate produced from the ion-conductingmaterial. The term “plate” is to be interpreted widely in thisconnection. It may also be a sheet or a layer applied to a support. Thefirst plate may be put on a second plate which forms the base, and thefirst plate may produce the walls of the containers. The walls may beformed for example by the inner wall of perforations formed in the firstplate. The second plate may be produced for example from glass or atransparent plastic. According to a further embodiment feature, ahydrophobic covering layer is applied to the first plate. Thisfacilitates the filling of the containers. The hydrophobic material maybe formed from a sheet which is expediently formed from an opaquematerial.

[0025] The ion-conducting material may be provided between twoelectrodes. The electrodes may be provided separately from thecontainers. The electrodes may be produced from conventional materialsuch as, for example, silver, gold, platinum, copper, aluminum orelectrically conducting plastic and the like. They may, for example, beattached to the ion-conducting material or be in ion-conducting contacttherewith via an aqueous solution which permeates the ion-conductingmaterial.

[0026] Exemplary embodiments of the invention are explained in moredetail below by means of the drawings. These show:

[0027]FIG. 1 a diagrammatic cross-sectional view of a first microtiterplate,

[0028]FIG. 2 a diagrammatic cross-sectional view of a second microtiterplate,

[0029]FIG. 3 a diagrammatic cross-sectional view of a third microtiterplate,

[0030]FIG. 4 a diagrammatic cross-sectional view of a fourth microtiterplate,

[0031]FIG. 5 a diagrammatic cross-sectional view of a fifth microtiterplate,

[0032]FIG. 6 a view from above onto the microtiter plate shown in FIG.4,

[0033]FIG. 7a to c a first method variant,

[0034]FIG. 7d to f a modification of the first method variant,

[0035]FIG. 8a to c a second method variant,

[0036]FIG. 9a to c a third method variant,

[0037]FIG. 10a to c a fourth method variant,

[0038]FIG. 11a to c a fifth method variant,

[0039]FIG. 12a to c a sixth method variant,

[0040]FIG. 13 a fluorimetric evaluation using a poly-acrylamidemicrotiter plate and

[0041]FIG. 14 a fluorimetric evaluation using an agarose microtiterplate.

[0042]FIG. 1 shows a diagrammatic cross-sectional view of a firstmicrotiter plate with a particularly simple design. This comprises afirst plate 1 which has on one side a plurality of recesses 2. Each ofthe recesses 2 forms a container B for receiving a solution containingan analyte. The first plate 1 may consist for example of an agarose orpolyacrylamide gel. Such gels can for example be cast in the form ofconventional microtiter plates. It is also possible to produce the firstplate 1 from ion-conducting material such as cellulose or derivativesthereof. The shaping can in this case take place by means ofcompression. The first plate 1 may moreover be produced from porouspolystyrene, polyvenyl chloride, polyethylene, polycarbonate, polymethylmetacrylate, polypropylene and the like. It is possible in this case forthe microtiter plate to be produced by the injection molding method.Reference number 3 designates the electrodes attached on the transversesides of the first plate 1. The electrodes 3 can be produced from metalssuitable for this purpose, such as platinum, gold, silver, anelectrically conducting plastic and the like.

[0043] In the second microtiter plate shown in FIG. 2, the first plate 1which is produced from ion-conducting material is placed on a secondplate 4. The first plate 1 has in this case a plurality of perforations5. The inner walls of the perforations 5 form the walls of thecontainers B. The base Bo thereof is formed by the side, facing thefirst plate 1, of the second plate 4. The second plate 4 can be producedfor example from glass, quartz or polystyrene. It is expedientlydesigned to be transparent. The second microtiter plate can be producedin a simple manner by casting an agarose gel for example on a glassplate. The perforations in the agarose gel can be produced by suitableplastic cores which have been previously placed on the glass plate andare removed again after solidification of the agarose or polyacrylamidegel.

[0044] For example detection entities such as peptites, proteins,nucleic acids and the like can be immobilized on the base Bo. It is alsopossible for the base Bo to be activated by chemical groups presentthereon, such as aldehyde, epoxide, amino groups or biotin. The base Bomay, however, also be produced from an ion-conducting material.

[0045] In the third, fourth and fifth microtiter plates shown in FIGS. 3to 5, in each case a hydrophobic covering layer 7 is applied to thefirst plate 1. The hydro-phobic covering layer 7 has two perforations 8which correspond to the first perforations 5. In the fourth microtiterplate shown in FIG. 4, the first plate 1 has neither first recesses 2nor first perforations 5. The first plate 1 in this case forms the baseBo of the containers B. In the exemplary embodiment shown in FIG. 5,first recesses 2 are provided in the first plate 1 and correspond to thesecond perforations 8 in the covering layer 7. The first perforations 5correspond to the second recesses 6, so that recesses 6 form the lowerpart, i.e. a lower wall section and the base Bo, of the containers B.

[0046]FIG. 6 shows once again in a view from above the three-layerstructure of the microtiter plate of the invention shown in crosssection in FIGS. 3 to 5. The recesses 2, and first 5 and secondperforations 8, can of course also be designed to be rectangular orsquare.

[0047] A subregion of the container B is shown in FIGS. 7 to 12. Thecontainer has an opening Op at the top and is bounded by wall W and baseBo. The walls W and/or the base Bo consist of an ion-permeable material.This material has the property, on contact with an electro-lyte and onapplication of a voltage, of permitting an electrophoretic ion flux intothe material. M designates the region of the container B which isencompassed by an optical measurement. The region M may include thebase. The region M may be, for example, a central region of thecontainer B.

[0048] In the first method variant shown in FIG. 7a to c, a detectionentity N is bound to the base Bo. This may be, for example, a substratefor proteases or other lytic proteins. The detection entity N may belabeled for example by means of a fluorophore. An analyte is designatedwith the reference letter A. This may be for example a protease or otherlytic proteins. On addition of the analyte A, the detection entity N iscleaved by the analyte and thus a fragment Fr is liberated. Onapplication of an electric field (FIG. 7c), an electrophoretic forceacts on the fragment Fr and the fragment Fr migrates into or onto thewall W of the container B. The concentration of the fragment Fr in thecontainer B decreases. The fragment Fr has a label, e.g. a fluorophore,or can be detected optically because of other properties. Theconcentration of the fragment can be determined by means of afluorescence measurement in the region M. In this arrangement, thedecrease in fluoresence indicates the present of the analyte A to bedetected.

[0049]FIG. 7d to 7 f depicts a homogeneous method variant of the methoddescribed in FIG. 7a to c. A charged and freely movable detection entityN is present in a container B. The detection entity N is charged and maybe labeled for example by means of a fluorophore. The detection entitymay be, for example, a substrate for proteases or other lytic proteins.An analyte A is brought into contact with the detection entity N. Thismay be for example a protease or other lytic proteins. On addition ofthe analyte A, the detection entity N is cleaved by the analyte A intotwo fragments Fr1 and Fr2. Fr1 is uncharged and can be detectedoptically for example through a fluorophore. Fr2 is charged. Onapplication of an electric field (FIG. 7f) an electro-phoretic forceacts on the detection entity N and the charged fragment Fr2. Detectionentity N and fragment Fr2 migrate into or onto the wall W of thecontainer B. The uncharged fragment Fr1 remains in the container B. Theconcentration of the fragment Fr1 in the container is an indicator ofthe presence of the analyte A. The concentration of the fragment Fr canbe determined by an optical measurement, for example a fluoresencemeasurement in the region M.

[0050] In the second method variant shown in FIG. 8a to c, a detectionentity N is bound to the base Bo of the container B. This is, forexample, a ligand, an antigen, a receptor, an antibody or a nucleicacid. An analyte A is brought into contact with the detection entity Nin a solution. The analyte A may be, for example, a receptor, anantibody, a ligand, an antigen or complementary nucleic acid. Theanalyte A binds to the detection entity N. The analyte A is labeled forexample by means of a fluorogen F. On application of an electric field,the analyte A migrates into the ion-conducting wall W. If the detectionentity N is specific for the analyte A, part of the analyte A binds tothe detection entity N. The binding of the analyte A to the seconddetection entity N can be detected by means of a fluorescence signal dueto the fluorophore F.

[0051] In the third method variant shown in FIG. 9a to c, an analyte Adisplaces a detection entity N from its binding to a defined bindingsite. The binding of the analyte A and the binding of the detectionentity N is specific in each case. The binding site may be, for example,an antibody immobilized on the base Bo or an immobilized nucleic acid.The detection entity N may be a specific antigen or a complementarynucleic acid. Displacement of the detection entity N by the analyte Aresults in free mobility of the previously immobilized detection entityN. The detection entity N is charged. Application of an electric fieldmoves the detection entity N into or onto the ion-permeable wall W. Theconcentration of the detection entity N is found by an opticalmeasurement in the region M. The decrease, observable in this case, of apreviously present fluorescence signal is specific for the presence ofthe analyte A in the solution.

[0052] In the fourth method variant shown in FIG. 10a to c, an analyte Aand a charged and freely mobile detection entity N is present in thecontainer B. The analyte A is labeled for example by means of afluorophore. On addition of the analyte A there is a binding, specificfor analyte A, with the detection entity N. Application of an electricfield moves the charged detection entity N and the analyte A boundthereto into or onto the ion-permeable wall W outside the region M. Thedecrease in the concentration of the fluorescent analyte A in thecontainer B can be detected by fluorimetry in the region M.

[0053] In the fifth method variant shown in FIG. 11a to c, a positivelycharged analyte A and a negatively charged detection entity N is presentin the solution. The analyte A is labeled for example by means of afluorophore. On addition of the analyte A there is a binding, specificfor analyte A, with the detection entity N. Binding of the analyte A tothe detection entity N makes the total charge of the complex zero. Onapplication of an electric field eF, the uncharged complex of detectionentity N and analyte A bound thereto remains in the container B. Unboundanalyte A is moved into or onto the ion-permeable wall W. The diminisheddecrease in the concentration of the fluorescent analyte A in thecontainer B compared with a sample without detection entity N can bedetected by fluorimetry in the region M. The diminished decreaseindicates the presence of the analyte A.

[0054] In the sixth method variant shown in FIG. 12a to c, the base Boof the container B is ion-permeable. In addition, a first detectionentity N is bound in the base Bo. The base Bo may consist for example ofactivated agarose or polyacrylamide to which antibodies or nucleic acidshave been-bound as detection entity N. A charged analyte A is introducedinto the container B.

[0055] The analyte A may be labeled for example by means of fluorophore.The analyte A comes into contact with detection entity N throughdiffusion or through application of an electric field eF. The contactresults in a specific binding between analyte A and detection entity N.Application of an electric field eF moves unbound analyte A out ofregion M, which is detected in an optical measurement. Only the boundanalyte A is detected in an optical measurement of the region M. Thefluorescence in region M is an indicator of the presence of the analyteA to be detected.

[0056]FIG. 13 shows a fluorimetric evaluation using a microtiter plateof the invention produced from polyacrylamide. A microtiter platefluorescence reader is used for the evaluation. Immobilizedoligonucleotide A of sequence 5′-TAA CAC AAC TGG TGT GCT CCT GGA-3′ (SEQID NO: 1) was present in all the samples 1 to 12. Samples 1 to 4 are incontact with TAMRA-labeled oligonucleotide A′ of sequence 5′-TAMRA-GAGCTA GGA CCT CTT CTG TCC AGG AGC ACA CCA GTT GTG TAA-3′ (TAMRA-SEQ ID NO:2) which is complementary at its 3′ end to the immobilizedoligonucleotide A. Samples Nos. 5 to 8 are in contact with TAMRA-labeledoligonucleotide K of sequence 5′-TAMRA-TAG GGT CAA TGC CAC CCT TTT AACCTA TCC GGA TTT ACG-3′ (5′-TAMRA-SEQ ID NO: 3), which is notcomplementary to oligonucleotide A. Samples Nos. 9 to 12 merelycontained buffer. The fluorescence is shown here in arbitrary units.

[0057]FIG. 14 shows the results of a fluorimetry evaluation using amicrotiter plate of the invention produced from agarose. TAMRA-labeledoligonucleotide A′ was present in all the samples. Samples Nos. 1 to 4are in contact with RecA protein. Samples Nos. 9 to 12 contained onlybuffer. The fluorescence is shown in arbitrary units.

[0058] The detection can be carried out considerably more quickly byapplying an electric field. The fluorescence can be detected immediatelyin the predetermined region of the container. The region may be acentral region of the container, a region in the vicinity of a wall, orthe base of the container.

EXAMPLES

[0059] 1. Production of a Supported, Ion-Conductive,Polyacrylamide-Based Microtiter Plate With Detection Entities.

[0060] A cassette consisting of two glass plates and 1 mm Teflon spacerswith the internal volume 1 mm×80 mm×120 mm was provided on the innersides of each of the glass plates with a plastic support (Gelbond PAGFilm, Pharmacia-Amersham), which are suitable for covalent coupling toacrylamide. A plastic support was provided with rows and columns ofsquare recesses which were each 2 mm×2 mm in size and were each 9 mmapart. The recesses were produced with the aid of a cutting blotter(Graphtec). A solution of 20% acrylamide with a monomer to crosslinkerratio of 29:1 was prepared in 1×TBE buffer. Acrydite oligonucleotides A(Eurogentec, Belgium) were added in a concentration of 10 μmol/l asdetection entity before the polymerization. To start the polymerization,70 μl of 10% (w/v) fresh ammonium persulfate and 20 μl of TEMED wasadded, and the solution was poured into the cassette. After one hour,the glass plates were removed and the microtiter plate was placed in aslab electrophoresis chamber with the plastic support with the recess ontop. Unbound charged constituents were removed by subjecting themicrotiter plate to an electrophoresis in 1×TBE at 100 V for one hour.

[0061] 2. Procedure for a DNA Hybridization Assay Using thePolyacrylamide-Based Microtiter Plate

[0062] 2 μl portions of sample solution in 1×TBE buffer were put intothe recess of the microtiter plate. Four sample solutions in each casecontained:

[0063] a) 10 pmol of the TAMRA-labeled oligonucleotide A′ prepared byTibMolbiol, Berlin, with the sequence complementary to oligonucleotideA.

[0064] b) 10 pmol of the TAMRA-labeled oligonucleotide K, prepared byTibMolbiol, Berlin, and

[0065] c) no oligonucleotide

[0066] Oligonucleotide A′ and K were labeled at the 5′ end with afluorophore (TAMRA). The microtiter plates were brought into contactwith TBE buffer on the sides facing the electrodes, and the microtiterplate was subjected to an electrophoresis at 50 V for 10 minutes.

[0067] 3. Fluorimetric Evaluation of the DNA Hybridization Assay

[0068] The microtiter plate was inserted into a microtiter plate reader(Lambda 320, MWG-Biotech), and the fluorescence of the recess wasmeasured. The fluorescence was determined with excitation at 540 mm andemission at 590 nm in reflected light mode. The fluorescence in thesamples with oligonucleotide A′ was distinctly increased compared withthe fluorescence of the samples with oligonucleotide B and the pure TBEsample. The increased fluorescence of the sample with oligonucleotide A′indicates the specific binding of oligonucleotide A′ to the Acryditeoligonucleotide A.

[0069] 4. Production of a Supported, Ion-Conductive, Agarose-BasedMicrotiter Plate

[0070] Two combs each with four shaping cores of Teflon were suspended 9mm apart 1 mm above the base in a chamber consisting of a glass platewith tightly surrounding rims of dimension 80 mm×40 mm. The edge lengthof the square shaping cores was 2 mm×2 mm in each case. The shapingcores were 9 mm apart. A 2% agarose suspension in TBE buffer was heatedin a microwave until the agarose was completely swollen, and about 12 mlof the suspension was poured into the chamber. After the agarose hadsolidified, the shaping cores were removed. An arrangement of 2×4recesses with a square opening of 2 mm×2 mm and a depth of about 3 mmhad been produced by means of the shaping cores in the producedmicrotiter plate. An opaque plastic sheet with recesses was placed onthe solidified agarose in such a way that the recess in the sheetcoincided with the recesses in the agarose.

[0071] 5. Procedure for a DNA-Protein Binding Assay Using theAgarose-Based Microtiter Plate.

[0072] 8 μl of a solution of TBE, 10 mmol/l MgCl₂ and, as detectionentity, 10 μmol/l oligonucleotide K were introduced into each of theeight recesses in the microtiter plate. Oligonucleotide K was providedwith a fluorophore (TAMRA) at the 5′ end for labeling. 4 μl of RecAprotein (Roche) of a concentration of 1 μg/μl in RecA buffer (10 mmol/lTris Cl, 10 mmol/l MgCl₂, 1 mmol/l DTT, pH 8) was added as sample intoeach of four recesses. The same volume of RecA buffer without RecAprotein was introduced as negative control into the remaining fourrecesses. The microtiter plate was incubated at 37° C. for 30 min andthen exposed to an electrophoresis at 50 V for 10 min.

[0073] 6. Fluorimetric Evaluation of the Protein-DNA Binding Assay

[0074] The microtiter plate was inserted into a microtiter plate reader(Lambda 320, MWG-Biotech) and the fluorescence was measured in theregion of the recess. The fluorescence was determined with excitation at540 mm and emission at 590 nm in reflected light mode. The fluorescencein the samples with RecA protein was distinctly increased compared withthe fluorescence of the samples without RecA protein. The increasedfluorescence of the sample with RecA protein indicates the change in theelectrophoretic mobility of binding of oligonucleotide K owing to thebinding of RecA protein to oligonucleotide K.

List of reference symbols

[0075]1 first plate

[0076]2 first recess

[0077]3 electrode

[0078]4 second plate

[0079]5 first perforation

[0080]6 second recess

[0081]7 hydrophobic covering layer

[0082]8 second perforation

[0083] B container

[0084] Bo base

[0085] A analyte

[0086] N detection entity

[0087] eF electric field

[0088] M region of the container whose optical change is detected

[0089] W wall

1 3 1 24 DNA Artificial sequence Synthetic oligonucleotide 1 taacacaactggtgtgctcc tgga 24 2 42 DNA Artificial sequence Syntheticoligonucleotide 2 gagctaggac ctcttctgtc caggagcaca ccagttgtgt ta 42 3 39DNA Artificial sequence Synthetic oligonucleotide 3 tagggtcaatgccacccttt taacctatcc ggatttacg 39

1. A method for detecting an analyte (A) in a liquid having thefollowing steps: (a) providing a solution comprising a detection reagent(N) in a container (B), (b) adding the analyte (A) to the solution, (c)applying an electric field (eF) acting on the solution by means ofelectrodes located outside the container (B), so that a concentration ofan entity which is specific for the presence of the analyte (A) changesin a region (M) of the container (B), and (d) optically detecting theconcentration change.
 2. The method as claimed in claim 1, where thesolution is incubated after step b.
 3. The method as claimed in claim 1,where the container (B) consists at least sectionally of anion-conducting material, and the electric field (eF) is applied in stepc in such a way that a migration of ions in the material is broughtabout thereby.
 4. The method as claimed in claim 1, where the opticaldetection of the concentration change takes place through an opening(Op) and/or a base (Bo) of the container (B).
 5. The method as claimedin claim 1, where the entity is the detection reagent (N), a reactionproduct formed from the detection reagent (N) and the analyte (A), or acompetitor.
 6. The method as claimed in claim 1, where the detectionreagent (N) comprises a receptor, a competitor, or a precursor of thereaction product.
 7. The method as claimed in claim 6, where thereceptor is selected from the following group: peptide, protein, nucleicacid, sugar, antibody, lectin, avidin, streptavidin, PNA or LNA.
 8. Themethod as claimed in claim 1, where the entity is labeled with afluorophore.
 9. The method as claimed in claim 1, where the entity isbound onto and/or in a base (Bo) of the container (B).
 10. The method asclaimed in claim 1, where, for optical detection of the concentrationchange, a light beam is passed at least through the region (M), and thechange thereof brought about the entity is measured.
 11. The method asclaimed in claim 10, where the light beam is guided so that it enters oremerges substantially perpendicularly from a base (Bo) or an opening(Op) of the container (B).
 12. The method as claimed in claim 1, wherethe electric field (eF) is applied simultaneously across a plurality ofcontainers (B) arranged in succession.
 13. The method as claimed inclaim 12, where the optical detection of the concentration change takesplace simultaneously in the plurality of containers (B).
 14. Amicrotiter plate having a plurality of containers (B) for receiving aliquid, characterized in that the containers (B) are formed at leastsectionally from an ion-conducting material, wherein the containers (B)comprise a base (Bo) and walls (W).
 15. The microtiter plate as claimedin claim 14, where the walls (W) and/or the base (Bo) of the containers(B) are produced from the ion-conducting material.
 16. The microtiterplate as claimed in claim 15, where the base (Bo) is produced from anelectrical insulator.
 17. The microtiter plate as claimed in claim 14,where the base (Bo) is activated for the binding of a ligand, receptor,or substrate.
 18. The microtiter plate as claimed in claim 14, where areceptor or a substrate is immobilized on the base (Bo).
 19. Themicrotiter plate as claimed in claim 18, where different receptors areimmobilized on predetermined sections of the base (Bo).
 20. Themicrotiter plate as claimed in claim 14, where the base (Bo) is producedfrom glass, quartz, or plastic.
 21. The microtiter plate as claimed inclaim 14, where the base (Bo) is produced from a transparent or opaquematerial.
 22. The microtiter plate as claimed in claim 14, where thewalls (W) of the containers (B) are produced from a porous material. 23.The microtiter plate as claimed in claim 14, where the walls (W) areactivated for binding a ligand.
 24. The microtiter plate as claimed inclaim 14, where the ion-conducting material is a material which isselected from the following group: agarose, polyacrylamide, cellulose,paper, paperboard, porous silicate, polystyrene, polyvinyl chloride,polycarbonate, nylon, polyethylene.
 25. The microtiter plate as claimedin claim 14, where the containers (B) have a substantially rectangularcross section.
 26. The microtiter plate as claimed in claim 14, wherethe containers (B) are designed in the form of recesses (2, 5) in afirst plate (1) produced from the ion-conducting material.
 27. Themicrotiter plate as claimed in claim 26, where the first plate (1) isput onto a second plate (4) which forms the base (Bo), and the firstplate (1) produces the walls of the containers (B).
 28. The microtiterplate as claimed in claim 26, where a hydrophobic covering layer (7) isapplied to the first plate (1).
 29. The microtiter plate as claimed inclaim 28, where the hydrophobic covering layer (7) is formed from asheet.
 30. The microtiter plate as claimed in claim 29, where the sheetis formed from an opaque material.
 31. The microtiter plate as claimedin claim 14, where the ion-conducting material is provided between twoelectrodes (3).
 32. The microtiter plate as claimed in claim 31, wherethe electrodes (3) are arranged parallel to two mutually opposite walls(W) of the container (B).
 33. The microtiter plate as claimed in claim31, where the electrodes are produced from silver, gold, platinum,copper, aluminum or electrically conductive plastic.